Sample Processing Methods

ABSTRACT

A method of processing a sample may include introducing a sample into a vessel, the vessel having proximal and distal ends, the sample being introduced into the proximal end of the vessel; incubating the sample in the vessel with a substance capable of specific binding to a preselected component of the sample; propelling components of the incubated sample, other than the preselected component, toward the proximal end of the vessel by clamping the vessel distal to the incubated sample and compressing the vessel where the incubated sample is contained; propelling the preselected component toward a distal segment of the vessel by clamping the vessel proximal to the preselected component and compressing the vessel where the preselected component is contained; and mixing the preselected component with a reagent in the distal segment of the vessel.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/782,354, filed May 18, 2010, which is a division of U.S. applicationSer. No. 10/773,775, filed Feb. 5, 2004 (now issued U.S. Pat. No.7,718,421), which claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/445,304, filed Feb. 5, 2003, the entiredisclosures of which are hereby incorporated herein by reference intheir entirety. The following U.S. Patent Applications are also herebyincorporated herein by reference in their entireties: Ser. No.09/910,233 (now issued U.S. Pat. No. 6,748,332); Ser. No. 09/782,732(now issued U.S. Pat. No. 6,780,617); and Ser. No. 10/241,816 (nowissued U.S. Pat. No. 7,799,521).

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

“This invention was made with Government support under grant numbers2R44HL67568-02, 1R43AI55079-01, and 1R43HL074689-01 awarded by theNational Institutes of Health and contract number DAAD13-03-C-0086awarded by the Department of Defense. The Government has certain rightsin the invention.” This statement is included solely to comply with 37C.F.R. §401.14(a)(f)(4) and should not be taken as an assertion oradmission that the application discloses and/or claims only oneinvention.

INTRODUCTION

Sample preparation is frequently required in performing diagnosticassays, particularly in the processing of biological samples. Abiological sample, for instance, typically undergoes intensive,demanding processing before it is in condition suitable for an assay.Proper sample preparation often requires precise conditions, such asparticular temperatures, concentrations, reagent volumes, and,especially, the removal of materials that can interfere with the desiredassay. Frequently a raw sample must be removed to a distant location toreceive proper processing by highly skilled personnel in a tightlycontrolled laboratory setting. Conventional processing devices andmethods often require large, highly complex and sophisticatedinstrumentation. These factors of conventional sample processingnecessarily cause a delay in the time to result, high costs, compromisedsample integrity and limitations on the practicality of using diagnosticassays in many instances.

SUMMARY

The present disclosure provides devices and methods for processingsamples. The disclosed devices and methods can facilitate thepreparation of samples through multiple processing steps.

In one aspect, a sample processing tubule may include a first segment, asecond segment, and a third segment. Each segment may be defined by thetubule, may be fluidly isolated, at least in part by a breakable seal,may be so expandable as to receive a volume of fluid expelled fromanother segment, and may be so compressible as to contain substantiallyno fluid when so compressed. Each segment may contain at least onereagent.

In another aspect, a method of processing a sample may includeintroducing a sample into a tubule discretized by breakable seals into aplurality of fluidly isolated segments, wherein the tubule has aproximal end for receiving waste and a distal end for conducting anassay; incubating the sample in a segment of the tubule with a substancecapable of specific binding to a preselected component of the sample;removing waste from the preselected component by clamping the tubuledistally of the segment containing the preselected component andcompressing that segment; and releasing a reagent to mix with thepreselected component from a fluidly isolated adjacent distal segment bycompressing at least one of the segment containing the preselectedcomponent and a segment containing a reagent distal of that segment,thereby opening a breakable seal and either propelling the reagent intothe segment containing the preselected component or propelling thepreselected component into the segment containing the reagent.

The disclosed devices and methods can provide significant advantagesover the existing art. In certain embodiments, a tubule may beprepackaged with reagents for a desired sample processing protocol,thereby providing the materials for an entire assay in one convenientpackage. In certain embodiments, waste products are segregated from atarget of interest early in the processing, so that the processed sampledoes not come into contact with surfaces that have been touched by theunprocessed sample. Consequently, trace amounts of reaction inhibitorspresent in the unprocessed sample that might coat the walls of thetubule are less likely to contaminate the processed sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a front elevation view of an exemplary embodiment of a sampletube including a tubule. FIG. 1B is a cross sectional view of a sampletube positioned inside an analyzer.

FIG. 2A is a cross sectional view of a sample tube including a tubule.FIG. 2B is a perspective view of another exemplary embodiment of asample tube.

FIGS. 3A-B are, respectively, front and side elevation views of anexemplary embodiment of a sample tubule.

FIG. 4A is a cross sectional view of an exemplary embodiment of a sampletube positioned in an analyzer. FIG. 4B is a schematic close-up view ofan embodiment of a biological sample.

FIGS. 5A-B are, respectively, cross sectional and perspective views ofexemplary embodiments of sample tubes positioned in analyzers.

FIGS. 6A-C are cross sectional views of an embodiment of a samplecollection device receiving a sample.

FIGS. 7A-B are, respectively, cross sectional and perspective views ofexemplary embodiments of grinding systems.

FIGS. 8-10 are graphs of experimental data generated using selectedexemplary embodiments of the disclosed devices and methods.

DETAILED DESCRIPTION

The present disclosure describes devices and methods for processingsamples. In several embodiments, segmented tubules provide a convenientvessel for receiving, storing, processing, and/or analyzing a biologicalsample. In certain embodiments, the segmented tubule facilitates sampleprocessing protocols involving multiple processing steps. In certainembodiments, a sample may be collected in a sample tubule, and thetubule then positioned in an analyzer; the analyzer may then manipulatethe tubule and its contents to process the sample.

A preferred embodiment includes a flexible tubule which has beensegmented into compartments by breakable seals. The individual segmentsmay contain various reagents and buffers for processing a sample. Clampsand actuators may be applied to the tubule in various combinations andwith various timings to direct the movement of fluid and to cause thebreakable seals to burst. This bursting of the breakable seals may leavean inner tubule surface that is substantially free of obstructions tofluid flow. In preferred embodiments, the flow of the biological samplemay be directed toward the distal end of the tubule as the processingprogresses, while the flow of waste may be forced to move in theopposite direction, toward the opening of the tubule where the samplewas initially input. This sample inlet can be sealed, possiblypermanently, by a cap with a locking mechanism, and a waste chamber maybe located in the cap to receive the waste for storage. A significantbenefit of this approach is that the processed sample does not come intocontact with surfaces that have been touched by the unprocessed sample.Consequently, trace amounts of reaction inhibitors present in theunprocessed sample that might coat the walls of the tubule are lesslikely to contaminate the processed sample.

In some embodiments the tubule may be so expandable as to be capable ofreceiving a volume of fluid from each of multiple segments in onesegment; this can allow sample and reagents to undergo certainprocessing steps in one segment leading to a simpler mechanicalstructure for performing assays. Another benefit of an embodiment usinga tubule that may be so expandable is that the same tubule structure maybe used to package different volumes of reagents within segments,allowing the same tubule to be packaged in differing ways depending uponthe assay to be performed.

The apparatus may include a transparent flexible tubule 10 (FIGS. 1A-B,FIGS. 2A-B, and FIGS. 3A-B) capable of being configured into a pluralityof segments, such as 16, 110, 120, 130, 140, 150, 160, 170, 180, and/or190, and being substantially flattened by compression. In an embodiment,a tubule may have at least two segments. In an embodiment, a tubule mayhave at least three segments. The flexible tubule can provideoperational functionality between approximately 2° C. and 105° C.,compatibility with samples, targets and reagents, low gas permeability,minimal fluorescence properties, and/or resilience during repeatedcompression and flexure cycles. The tubule may be made of a variety ofmaterials, examples of which include but are not limited to: polyolefinssuch as polypropylene or polyethylene, polyurethane, polyolefinco-polymers and/or other materials providing suitable characteristics.The tubule properties, such as transparency, wetting properties, surfacesmoothness, surface charge and thermal resilience, may affect theperformance of the tubule. These proprieties may be improved throughsuch exemplary processes as: seeding, plasma treating, addition ofadditives, and irradiation. In some embodiments, an additive materialmay be added to the plastic to improve selected characteristics. Forexample, a slip additive may be added, such as erucamide and/oroleamide; in some embodiment, a so-called “anti-block” additive may beadded. An additive may have a concentration in the plastic in the rangefrom about 0.01% to about 5.0%.

The tubule may be manufactured by a wide variety of suitable methodssuch as extrusion, injection-molding and blow-molding. In a preferredembodiment the tubule is continuously extruded. Alternative techniquesfor manufacturing the tubule include, e.g., casting, extruding orblowing films that can be fashioned by secondary processing operationsinto a suitable tubule. The tubule wall material may include multiplelayers by co-extrusion, or by film lamination. For example, an innerlayer may be chosen for high biocompatibility and an exterior layer maybe chosen for low gas permeability. As a further example, the interiorlayer may be readily formed into a breakable seal 14 (FIG. 2A-B andFIGS. 3A-B), such as a peelable seal, while the exterior layer may beresilient and highly impermeable. For use in the present disclosure itis preferred the tubule have a wall thickness of about 0.03 mm to about0.8 mm, preferably 0.03 mm to about 0.5 mm, with the tubule able to besubstantially flattened with an applied exterior pressure on the orderof 1 atmosphere.

In some embodiments, the apparatus may have toughened walls in at leastone segment to allow for the dislocation of clumps of cells from solidsample such as biopsy samples or solid environmental samples usinggrinding motions. An example of these toughened wall features, asillustrated in FIG. 7A, can be micro-teeth-like inner surfaces 109 onopposing faces of the tubule wall, which are offset such thatcompressing the tubule produces a sliding motion along the axis of thetubule. The tubule wall in the vicinity of these grinding surfaces 109may be fortified using reinforcement patches made of a suitablyresilient plastic such as polycarbonate or polyethylene terephthalate.The teeth-like inner surfaces may be made of similarly suitablematerials. In another embodiment, a pad, such as 214 illustrated inFIGS. 5A-B, having grinding surface feature can be attached on the innerwall of tubule. The pad can be made by toughened material, and thesurface feature can be created by using conventional mechanical,electrochemical or microelectromechanical methods, so that the pad canendure compression.

The sample tubule 10 may be partitioned into one or more segments 16,110, 120, 130, 140, 150, 160, 170, 180, and/or 190, and/or sub-segments18, 121, 122. In preferred embodiments, the segments are defined bybreakable seals 14 to fluidly isolate adjacent segments. This sealfeature can be useful in separating, for example, a dry reagent from aliquid reagent until the two can be reconstituted to perform a specificassay, or for separating chemically reactive species until the reactionis desired. As illustrated in FIGS. 3A-B, a breakable seal 14 may beformed in a region of the tubule 10 where opposing walls have beensubstantially joined, but not joined so strongly as to prevent the wallsfrom being later peeled apart without significantly marring the tubuleor the previously sealed surfaces. Such a seal may be termed a“peelable” seal. In a preferred embodiment, the peelable seal region maybe a band orthogonal to the axis of the tubule. It may span a tubulelength in the range of about 0.5 mm to 5 mm, preferably about 1 mm toabout 3 mm, most preferably about 1 mm. The seal preferably spans theentire width of the tubule so as to seal the segment. In someembodiments, the seal band may vary in height or shape and/or beoriented at an angle transverse to the axis of the tubule; suchvariations can change the peel characteristics.

Breakable seals 14 can be created between opposing walls of the tubuleby applying a controlled amount of energy to the tubule in the locationwhere the peelable seal is desired. For example, a temperaturecontrolled sealing head can press the tubule at a specific pressureagainst a fixed anvil for a specific time interval. Various combinationsof temperature, pressure and time may be selected to form a seal ofdesired size and peel-strength. Energy may be delivered, for example, bya temperature controlled sealing head maintained at a constanttemperature between 105° C. and 140° C. to heat a polypropylene tubingmaterial; an actuator capable of delivering a precise pressure between 3and 100 atmosphere over the desired seal region; and a control system todrive the sequencing of the actuator to a specific cycle time between 1and 30 seconds. Using this method, satisfactory seals have been createdin polypropylene tubules to peel open when subjected to an internalpressure on the order of 1 atmosphere. Alternate techniques to deliverthe sealing energy to the tubule include RF and ultrasonic welding.

In other embodiments, alternate tubule materials and blends of materialscan be used to optimize peelable seal performance. For example, twopolypropylene polymers of differing melting temperature can be blendedin a ratio such that the composition and melt characteristics areoptimized for peelable seal formation. In addition to or in lieu ofbreakable seals 14, the flexible tubule can further have one or morepressure gates 194, which are capable of reversibly opening and closingduring the operation of a test by applying a controlled force to asegment of the flexible tubule.

A filter can be embedded in a tubule segment. Examples of filters 206and 216 are shown in FIG. 4A and FIGS. 5A-B, respectively, In apreferred embodiment, a filter can be formed by stacking multiple layersof flexible filter material. The uppermost layer of the filter thatdirectly contacts a sample may have a pore size selected for filtration;the bottom layer of the filter may include a material with much largerpore size to provide a support structure for the uppermost layer when apressure is applied during filtration. In this preferred embodiment, thefilter may be folded to form a bag, with the edges of its open endfirmly attached to the tubule wall. The segment with the filter bag maybe capable of being substantially flattened by compressing the exteriorof the tubule.

In exemplary embodiments, one or more reagents can be stored either asdry substance and/or as liquid solutions in tubule segments. Inembodiments where reagents may be stored in dry format, liquid solutionscan be stored in adjoining segments to facilitate the reconstitution ofthe reagent solution. Examples of typical reagents include: lysisreagent, elution buffer, wash buffer, DNase inhibitor, RNase inhibitor,proteinase inhibitor, chelating agent, neutralizing reagent, chaotropicsalt solution, detergent, surfactant, anticoagulant, germinant solution,isopropanol, ethanol solution, antibody, nucleic acid probes, peptidenucleic acid probes, and phosphothioate nucleic acid probes. Inembodiments where one of the reagents is a chaotropic salt solution, apreferred component is guanidinium isocyanate or guanidiniumhydrochloride or a combination thereof. In some embodiments, the orderin which reagents may be stored in the tubule relative to the openingthrough which a sample is input, reflects the order in which thereagents can be used in methods utilizing the tube. In preferredembodiments, a reagent includes a substance capable of specific bindingto a preselected component of a sample. For example, a substance mayspecifically bind to nucleic acid, or a nucleic acid probe mayspecifically bind to nucleic acids having particular base sequences.

In other exemplary embodiments, a solid phase substrate can be containedwithin a tubule segment and used to capture one or more selectedcomponents of a sample (if such component is present in a sample), suchas a target microorganism or nucleic acids. Capturing can help to enrichthe target component and to remove reaction inhibitors from a sample.Substrates may be solid phase material which can capture target cells,virions, nucleic acids, or other selected components under definedchemical and temperature conditions, and may release the componentsunder different chemical and temperature conditions.

In some embodiments, a reagent can be coated on the substrate. Examplesof coatable reagent are: receptors, ligands, antibodies, antigens,nucleic acid probes, peptide nucleic acid probes, phosphothioate nucleicacid probes, bacteriophages, silica, chaotropic salts, proteinases,DNases, RNases, DNase inhibitors, RNase inhibitors, and germinantsolutions. In some embodiments, the substrate can be stored in a drysegment of the tubule while in other embodiments it can be storedimmersed in a liquid. In some embodiments, the order in which reagentsmay be stored in the tubule relative to the substrate and the openingthrough which a sample is input, reflects the order in which thereagents and the substrate can be used in methods utilizing theapparatus.

The substrate can be: beads, pads, filters, sheets, and/or a portion oftubule wall surface or a collection tool. In embodiments where thesubstrate is a plurality of beads, said beads can be: silica beads,magnetic beads, silica magnetic beads, glass beads, nitrocellulosecolloid beads, and magnetized nitrocellulose colloid beads. In someembodiments where the beads can be paramagnetic, the beads can becaptured by a magnetic field. Examples of reagents that may permit theselective adsorption of nucleic acid molecules to a functionalgroup-coated surface are described, for example, in U.S. Pat. Nos.5,705,628; 5,898,071; and 6,534,262, hereby incorporated herein byreference. Separation can be accomplished by manipulating the ionicstrength and polyalkylene glycol concentration of the solution toselectively precipitate, and reversibly adsorb, the nucleic acids to asolid phase surface.

When these solid phase surfaces are paramagnetic microparticles, themagnetic beads, to which the target nucleic acid molecules have beenadsorbed, can be washed under conditions that retain the nucleic acidsbut not other molecules. The nucleic acid molecules isolated throughthis process are suitable for: capillary electrophoresis, nucleotidesequencing, reverse transcription, cloning, transfection, transduction,microinjection of mammalian cells, gene therapy protocols, the in vitrosynthesis of RNA probes, cDNA library construction, and the polymerasechain reaction (PCR) amplification. Several companies offermagnetic-based purification systems, such as QIAGEN's MagAttract™,Cortex Biochem's MagaZorb™, Roche Applied Science's MagNA Pure LC™, andMagPrep® Silica from Merck & Co. All of these kits use negativelycharged particles and manipulate buffer conditions to selectively bind avariety of nucleic acids to the beads, wash the beads and elute thebeads in aqueous buffers. Many of the products used by these companiesuse chaotropic salts to aid in the precipitation of nucleic acids ontothe magnetic beads. Examples are described in U.S. Pat. Nos. 4,427,580;4,483,920; and 5,234,809, hereby incorporated herein by reference.

In some embodiments the substrate may be a pad 214 or 30 (FIGS. 5A-B,FIGS. 6A-C). In further embodiments, the substrate pad can include paper35, alternating layers of papers 34 with different hydrophobicproperties, glass fiber filters, or polycarbonate filters with definedpore sizes. In some embodiments, the pad may be a filter or impermeablesheet 38 for covering selected portion of the surfaces of the pad, saidfilter having a predetermined pore size. Such a filtration device can beused for separations of white blood cells 32 and red blood cells 33 (orother particles, such as virus or microorganisms) from whole blood 31and/or other samples. The pad 214 can be mounted on the tubule wall(FIGS. 5A-B) and/or on a sample collection tool 26. In some embodimentsthe pad can be soaked with a reagent solution while in other embodimentsit may be coated with dry reagents.

Preferred exemplary embodiments may include a linear arrangement of 2 ormore tubule segments 110, 120, 130, 140, 150, 160, 170, 180, and/or 190(FIG. 1B). A linear arrangement facilitates moving the sample andresultant waste and target through the tube in a controlled manner. Araw biological sample can be input through a first opening 12 (FIG. 2B)in a first segment 110 (FIG. 1B) of the tubule. Thereafter, waste from aprocessed sample can be moved back toward the first opening while thetarget is pushed towards the opposite end, thereby minimizingcontamination of the target by reaction inhibitors that may have becomeattached to the tubule wall, and confining the target to a clean segmentof the tubule which can contain suitable reagents for further operationsof the target. Some embodiments may use a plurality of at least threesegments, each containing at least one reagent. In some embodiments,these segments may contain reagents in the following order: the reagentin the second segment may be either a lysis reagent, a dilution or washbuffer, or a substrate; the reagent in the third segment may be either asubstrate, a lysis reagent, a washing buffer or a neutralizationreagent; the reagent in the fourth segment may be a wash buffer, asuspension buffer, an elution reagent, or nucleic acid amplification anddetection reagents. In some embodiments, the three segments may bearranged continuously, while in other embodiments, these three segmentsmay be separated by another segment or segments in between.

In some embodiments, a pressure gate 194 can be incorporated toselectively close and open a second opening, located at the distal endof the tubule, to collect the products generated during a test from thetubule for further processing, outside of the tubule. In someembodiments, this second opening may located in a segment 198 defined bytwo pressure gates 194 and 196 to store a product from the sampleprocessing segments. In some embodiments, a combination of a breakableseal and a pressure gate may be provided for transferring the contentsof the tubule to a second opening.

In some embodiments a tube closing device for closing the tube aftersample input may include a cap 20 (FIG. 1B) and/or clamp 310. Aninterface or adaptor 52 between the cap and the first opening of theflexible tubule may be used to ensure a secure, hermetic seal. In anexemplary embodiment, this interface may be threaded and may includetapered features 62 on the cap and/or a suitably rigid tube frame 50such that, when fastened together, the threads 64 can engage to mate thetapered features 62 between the tube frame and cap to provide a suitablelock. In this exemplary embodiment the cap may require ½ to 1 fullrotation to fully remove or attach from the tube holder. The combinationof thread pitch and taper angle in the joint can be selected to be botheasily manufactured and to provide feedback resistance to inform theuser that an effective seal has been created. In other embodiments thecap locking device may include snap fits, press fits, and/or other typesof “twist and lock” mechanism between the cap and tube holder, andsimilar arrangements in which the cap is permanently attached to thetubule, such as by hinging or tethering the cap.

Both the cap 20 and tube frame 50 can be made of a suitable injectionmolded plastic such as polypropylene. The tube frame 50 can, in turn, befastened to the flexible tube by a permanent, hermetic seal. Theexterior portion of the cap may be covered with ridges or finger gripsto facilitate its handling. Furthermore, the cap 20 may include an areafor attaching a sample identification mark or label. As a furtheralternative, the cap may be directly attached to the first openingflexible tube through a press fit or a collar that compresses theflexible tube opening against a protrusion in the cap to create ahermetic seal. The lock between the tube cap and tube holder may bekeyed or guided such that a collection tool 36 or features integratedinto the cap can be definitively oriented with respect to the tube tofacilitate sample processing and the flattening of the flexible tubule.Furthermore, the cap may incorporate features such as a ratchet orsimilar safety mechanism to prevent the cap from being removed after ithas been installed onto the opening of the flexible tube.

The cap 20 used to close the tubule in some embodiments may contain acavity 22 within it by making the cap body substantially hollow. In someembodiments, the hollow portion extends from the top of the cap body toan orifice at the base of the cap body. To form a chamber, the top ofthe cavity may be closed by fastening a cover onto the cap body. Thecover may be constructed of the same piece as the cap body. The covermay incorporate a vent hole 26 or may further incorporate an affixedmicrobe barrier, filter or a material that expands to close off the venthole when exposed to a liquid or specific temperature. The bottom of thechamber may be left open or closed by a breakable septum or valve. Thehollow chamber may further incorporate a flexible membrane or septum 24.This flexible septum could be manufactured using dip molding, liquidinjection silicone molding, blow molding, and/or other methods suitablefor the creation of thin elastomeric structures. The flexible septum canbe inserted into the cap body cavity 22 assembly so as to effectivelyisolate the interior portion of the tube from the exterior environmentafter the cap is in place on the tube. The flexible septum could bedesigned such that, in the absence of externally applied pressures, itsinherent stiffness ensures it is in a preferred, known state ofdeformation: As a further embodiment, the flexible septum may bereplaced by a plunger. In an exemplary embodiment, a cap bodyapproximately 30 mm high by 14 mm diameter may be injection molded of asuitable thermoplastic and contain an interior cavity having at least500 μL of available volume. The chamber in the cap body could be adaptedfor useful purposes such as holding or dispensing a reagent, serving asa reservoir to hold waste fluids, serving as a retraction space for anintegrated collection tool, or a combination of thereof.

The cap 20 may have an integrated collection tool 30 (FIG. 2B) such as aswab, capillary tube, liquid dropper, inoculation loop, syringe,absorbent pad, forceps, scoop or stick to facilitate the collection ofliquid and solid samples and their insertion into the tubule. Thecollection tool may be designed to collect and deposit a predeterminedamount of material into the tube. Reagents may be stored on thecollection tool itself. For example, the collection tool may include aswab impregnated with a dry salt such that when the swab is hydrated itwould suspend the salt off the swab into solution. Furthermore, thecollection tool and cap may be designed such that the collection toolportion retracts into the cap body after depositing the sample into thetubule to leave the tubule segments substantially unencumbered.

The chamber 22 in the cap may be fashioned to store a reagent. Toaccomplish this, for example, the base of the chamber may be closed by abreakable septum or valve (not shown) such that when the cap issqueezed, the septum breaks to release the reagent. Such a feature wouldbe useful, for example, if the cap were integrally formed with acollection tool such as a swab or stick. In this instance, the reagentreleased from the cap chamber could be used to wash a sample off thecollection tool into a tube segment or to lyse the sample contained onthe collection tool. Reagents may also be released from the cap chamberby opening the breakable septum using pressure generated by compressinga flexible tube segment to force fluid from the tube up into the capchamber. The chamber in the cap may be fashioned to store waste fluidsderived from processing within the tubule. In a preferred embodiment,the base of the chamber may be left open such that when connected to thefirst opening of the flexible tubule a fluid passage is formed betweenthe tubule and the chamber. As fluid is moved into the cap chamber, theflexible septum 24 contained within can move from an initial positionupward so as to accommodate the influx of new fluid. This septummovement can be facilitated by the incorporation of a vent hole 26 onthe cap body cover.

After fluid has been transferred into the cap chamber a clamp 310 oractuator 312 can act to compress the tubule and effectively seal off thecap chamber volume from the tubule segments. As an alternativeembodiment, the cap chamber may incorporate a pressure gate or checkvalve (not shown) to prohibit fluid flow from the cap chamber back intothe tube segments. As a further alternative, the flexible septum may beomitted with the cap chamber cover including a microbe barrier to permitthe free escape of contained gasses but retain all the liquid volumesand infectious agents in the tube. As a further alternative, theflexible septum can be replaced with a plunger that would move axiallyupward to accommodate additional fluid volumes transferred from the tubesegments to the cap chamber. Other methods to accommodate fluidic wastewithin the cap chamber can be readily envisioned without departing fromthe scope of the present disclosure.

A substantially rigid frame 50 may be provided to hold the flexibletubule 10 suitably taut by constraining at least the proximal and distalends of the tubule. In an exemplary embodiment, a first constraint maybe provided to permanently attach and seal the tubule to the framearound the first opening of the tube. This seal may be created bywelding the flexible tubule to the frame using thermal and/or ultrasonicsources. Alternatively, the seal may be created using a hot-meltadhesive joint with ethylene vinyl acetate, or by making a joint using aUV cure epoxy or other adhesives. In further embodiments, the tubule maybe mechanically sealed or insert-molded with the frame. A secondconstraint may be provided to attach and seal the tubule to the base ofthe frame. In an exemplary embodiment of this second constraint, thisend of the tubule may be sealed flat and attached to the rigid frame bythermal and/or ultrasonic welding techniques. Alternatively, this jointand seal may also be formed using adhesive or mechanical approaches. Inan alternative embodiment, the second seal may be similar to the firstseal, being substantially open to enable access to the contents of theflexible tubule from the second opening. The tubule and frame materialscan be optimized for joint manufacture. For example, the frame can bemade of polypropylene having a lower melting point than the thinnertubule to ensure more uniform melting across one or more weld zones. Tofacilitate welding between the tubule and the frame, the joint area maybe tapered or otherwise shaped to include energy directors or othercommonly used features enhance weld performance. In an exemplaryembodiment, the rigid frame can be made of any suitable plastic byinjection molding with its dimensions being approximately 150 mm tall by25 mm wide.

The rigid frame 50 can incorporate several features to facilitate thecompression and flattening of the flexible tubule. For example, in anexemplary embodiment, the flexible tubule 10 may be constrained only atits two axial extremities to allow maximum radial freedom to avoidencumbering the tubule's radial movement as it is compressed. In anotherembodiment, compression may be facilitated by including a relief area inthe frame, near the first opening of the tube. This relief area may beused to facilitate the flexible tubule's transition from a substantiallycompressed shape in the tubule segments to a substantially open shape atthe first opening. Other useful features of the rigid frame that canfacilitate flexible tubule compression may include an integral tubuletensioning mechanism. In an exemplary embodiment, this tension mechanismcould be manufactured by molding features such as cantilever or leaftype springs directly into the rigid frame to pull the tubule taut atone of its attachment points with the frame.

The rigid frame 50 can facilitate tube identification, handling, sampleloading and interfacing to the tube cap. For example, the frame canprovide additional area to identify the tube through labels or writing80 affixed thereto. The plastic materials of the frame may be colorcoded with the cap materials to help identify the apparatus and itsfunction. The frame may incorporate special features such as changes inthickness or keys to guide its orientation into a receiving instrumentor during manufacture. The frame may interface to a sleeve 90 orpackaging that covers or protects the flexible tubule from accidentalhandling damage, light exposure, and/or heat exposure. The body of therigid frame may also provide a convenient structure to hold the tube.The frame may have an integral collection tool 32 such as a deflector orscoop to facilitate sample collection into the apparatus. Thesample-receiving end of the frame may also incorporate a tapered orfunneled interior surface to guide collected sample into the flexibletube.

In some embodiments, a method of extracting nucleic acids frombiological samples by using the apparatus described in the previousparagraphs is contemplated. In certain embodiments, the sequence ofevents in such a test may include: 1) a biological sample collected witha collection tool, 2) a flexible tubule, which can include a pluralityof segments that may contain the reagents required during the test, andin which the collected sample can be placed using a first opening in thetubule, 3) at least one substrate that may be set at a controlledtemperature and/or other conditions to capture target organisms ornucleic acids during a set incubation period, 4) organisms or molecules,in the unprocessed sample, that may not bind to the substrate and couldthus be removed by transferring liquid to a waste reservoir, 5) storingwaste, in a waste reservoir, that can be segregated from the target by aclamp and/or actuator compressed against the tubule, 6) a wash buffer,released from another segment of the tubule, that can remove reactioninhibitors, 7) an elution reagent, from another segment, that canrelease the target bound to the substrate after incubation at acontrolled temperature, and 8) nucleic acids that can be detected bytechniques well known to those familiar in the art or collected througha second opening in the tubule. In exemplary embodiments the flow of thesample may be from the first opening towards the distal end of thetubule as the test progresses while the flow of waste may be towards theclosed sample input opening of the tubule, where a waste chamber in thecap of the tubule receives the waste for storage. Consequently,undesirable contact between a processed sample and surfaces in areaction vessel that have been touched by the unprocessed sample isavoided, thereby preventing reaction inhibition due to trace amounts ofreaction inhibitors present in the unprocessed sample and that mightcoat the walls of the reaction vessel.

Some embodiments may incorporate the use of a test tube 1, with aflexible tubule 10 divided into a plurality of segments, such assegments 16, 110, 120, 130, 140, 150, 160, 170, 180, and/or 190, thatmay be transverse to the longitudinal axis of the tubule, and which maycontain reagents, such as reagents 210, 221, 222, 230, 240, 250, 260,270, 280, and/or 290; as well as an analyzer, that may have a pluralityof actuators, such as actuators 312, 322, 332, 342, 352, 362, 372, 382,and/or 392, clamps, such as clamps 310, 320, 330, 340, 350, 360, 370,380, and/or 390, and blocks, for example 314, 344, and/or 394 (othersunnumbered for simplicity); opposing the actuators and clamps, toprocess a sample. Various combinations of these actuators, clamps,and/or blocks may be used to effectively clamp the tubule closed therebysegregating fluid. In exemplary embodiments, at least one of saidactuators or blocks may have a thermal control element to control thetemperature of a tubule segment for sample processing. The sampleprocessing apparatus can further have at least one magnetic field source430 capable of applying a magnetic field to a segment. The sampleprocessing apparatus can further have a detection device 492, such asphotometer or a CCD, to monitor a reaction taking place or completedwithin the tubule.

The combined use of the tube and the analyzer can enable many sampleprocessing operations. Collecting a sample, such as blood, saliva,serum, soil, tissue biopsy, stool or other solid or liquid samples, canbe accomplished by using a sample collection tool 30 that may beincorporated into the cap 20, or features 32 on the tube frame 50. Aftera suitable amount of the sample has been collected, the cap can beplaced onto the first opening of the tube to close the tube and depositthe sample into the first segment. Following this step, the samplecontained on the collection tool may be washed off or re-suspended withreagents contained in separate chambers within the cap by compressing apotion of the cap. The tube can then be loaded into the analyzer forfurther processing. Identification features, such as a barcode or an RFtag, can be present on the tube to designate the sample's identity in aformat that can be read by the analyzer and/or a user.

Opening a breakable seal of a tubule segment can be accomplished byapplying pressure to the flexible tubule to irreversibly separate thebound surfaces of the tubule wall. An actuator can be used to apply therequired pressure to compress a tubule segments containing fluid to opena breakable seal. In embodiments where a segment is delimited by twobreakable seals, A and B, the analyzer may preferentially break seal Aby physically protecting the seal B region with an actuator or clamp toprevent seal B from breaking while pressure is applied to the segment tobreak seal A. Alternatively, seal A may be preferentially opened byapplying pressure to the segment adjacent to seal A in a precise mannersuch that; seal A is first opened by the pressure created in theadjacent segment; after seal A is broken, the pressure between the twosegments drops substantially due to the additional, combined, segmentvolume; the reduced pressure in the combined segment is insufficient tobreak seal B. This method can be used to open breakable seals one at atime without using a protecting actuator and/or clamp. As a furtheralternative, the adherence of seal A may be inferior to that of seal Bsuch that seal A can break at a lower pressure than seal B.

A process of moving fluid from one segment to another segment mayinclude, for example, releasing a clamp on one end of the first segment,compressing a clamp on the other end of the first segment, releasing anactuator on the second segment, and compressing an actuator on the firstsegment to move the liquid from the first segment to the second segment.Alternatively, the clamp may be omitted or be opened after releasing theactuator on the second segment.

A process of mixing two substances, where at least one is liquid,located in adjacent segments may be accomplished by: releasing the clampbetween the two segments, moving the liquid contained in the firstsegment, through an opened breakable seal to the second segment; andalternatively compressing the second segment and the first segment toflow the liquid between the segments.

An agitation can be performed by alternatively compressing anddecompressing a tubule segment with an actuator, while both clamps thatflank the actuator are compressing the tubule. In another embodiment,agitation can be achieved by alternatively moving liquid between atleast two segments.

In embodiments where a tubule segment may contain a liquid having avolume exceeding the volume required for a protocol, a process ofadjusting the volume of the liquid in the segment can be executed by:compressing the tubule segment to reduce the gap of between the tubewalls to set the volume of the segment to a desired level and allowingthe exceeding liquid to flow to the adjacent segment, past a clamp atthe end of the segment or adjacent actuator; closing the tubule segmentwith the clamp or actuator, resulting in an adjusted volume of liquidremaining in the segment.

A process of removing air bubbles may include agitating a segmentcontaining the bubbly liquid. Another process of removing air bubblesmay include agitating a first segment containing liquid while closing asecond segment; opening the second segment and moving the liquid fromthe first segment to the second segment; agitating the second segmentand adjusting a position of the second actuator to move the liquid-airinterface near or above the upper end of the second segment, thenclamping the upper end of the second segment to form a fullyliquid-infused segment without air bubbles.

A dilution process can be conducted by using the liquid movement processwherein one of the segments includes a diluent and the other includes asubstance to be diluted.

A process of reconstituting a reagent from dry and liquid componentsseparately stored in different tubule segments or sub-segments mayinclude compressing the tubule segment or sub-segment containing theliquid components to open the breakable seal connecting to the dryreagent segment, moving the liquid into the dry reagent segment orsub-segment, and mixing the dry reagent and liquid components using themixing process.

Filtration can be performed by using a filter 206 (FIG. 4A) positionedbetween two segments or two sub-segments. For example, a whole bloodsample can be deposited into a first segment with a filter bag. A poresize of the filter can be selected for blood cell filtration. A clamp300 can then close the end of the segment opposite to the filter bag,and an actuator 302 can compress the first segment to generate pressureto drive plasma flow through the filter into a second segment. Inanother embodiment, a coagulation, aggregation or agglutination reagent,such as antibody 204 against red cell 202 surface antigens, a red cellcoagulate, can be used to induce red cell-red cell binding to formclusters prior to the filtration. The pore size of the filter can beselected to block the clusters while allowing non-aggregated cells toflow through. Applying pressure on the first segment containing red cellclusters and blood can enrich the white cells 208 in the second segment.

In some embodiments, a grinding process can be conducted by using anactuator to alternately compress and decompress a tubule segment havinga toughened wall with a micro-teeth-like inner surface 109 (FIG. 7A),and thus break-up a solid sample, such as biopsy tissue sample, withinthe tubule segment. In another embodiment, small glass beads can be usedwith the solid sample to improve the performance of grinding. In afurther embodiment, a grinding wheel 450 driven by a motor 452 can beused to form a rotational grinding onto the sample in the tubule segmentand drive the movement of glass beads and a biological sample 200 toimprove grinding performance. The temperature of a liquid reactant inthe segment can be selected so as to improve the grinding result.

Incubation of the contents in a segment can be achieved by setting thecorresponding actuator and/or block temperature and applying pressure tothe segment to ensure a sufficient surface contact between the tubulewall of the segment and the actuator and the block, and bring thecontents of the tubule segment to substantially the same temperature asthe surrounding actuator and/or block temperature. The incubation can beconducted in all processing conditions as long as the temperatures ofall involved segments are set as required.

Rapid temperature ramping for incubation can be achieved by incubating afluid in a first segment at a first temperature and setting a secondtemperature for a second segment adjoining the first segment, afterincubation at the first temperature is finished, liquid is rapidly movedfrom the first segment to the second segment and incubated at the secondtemperature.

A flow driving through a flow-channel process can be performed bycompressing the tubule with a centrally-positioned actuator, and itsflanking clamps if any, to form a thin-layer flow channel with a gap ofabout 1 to about 500 μm, preferably about 5 to about 500 μm throughsegment. The adjacent actuators compress gently on the adjacent segmentsin liquid communication with the flow-channel to generate an offsetinner pressure to ensure a substantially uniform gap of the thin-layerflow channel. The two flanking actuators can then alternatively compressand release pressure on the tubule on their respective segments togenerate flow at controlled flow rate. Optional flow, pressure, and/orforce sensors may be incorporated to enable closed-loop control of theflow behavior. The flow-channel process can be used in washing,enhancing the substrate binding efficiency, and detection.

A magnetic bead immobilization and re-suspension process can be used toseparate the beads from the sample liquid. The magnetic field generatedby a magnetic source 430 (FIG. 1B) may be applied to a segment 130containing a magnetic bead suspension 230 to capture and immobilize thebeads to the tube wall. An agitation process can be used during thecapturing process. In another embodiment, a flow-channel can be formedon the segment with the applied magnetic field, and magnetic beads canbe captured under flow to increase the capturing efficiency. Forre-suspending immobilized beads, the magnetic field may be turned off orremoved, and an agitation or flow-channel process can be used forre-suspension.

A washing process to remove residual debris and reaction inhibitors froma substrate may be conducted by using three basic steps: First anactuator can compress a segment containing the substrate, such asimmobilized beads or a sheet, to substantially remove the liquid fromthis segment. Second, a washing buffer may be moved to the segment byusing a process similar to that of reconstituting a reagent from dry andliquid components. For bead-based substrates, a bead re-suspensionprocess can be used followed by bead re-capture on the tubule wall.Third, after a mixing or agitation process, the actuator can compressthe segment to remove the used wash liquid from the segment. In anotherembodiment, a flow-channel can be fowled in the segment containing asubstrate, which may be either immobilized beads or a sheet. Aunidirectional flow wash, having laminar characteristics, is generatedthrough the flow channel with the substrate. Finally, all the actuatorsand clamps, if any, can be closed to remove substantially all the liquidfrom the segments. In a further embodiment, a combination of thedilution based washing and the laminar flow based washing can be used tofurther enhance the washing efficiency.

Lysis can be achieved by heating a sample at a set temperature or byusing a combination of heat and chemical agents to break open cellmembranes, cell walls or uncoat virus particles. In another embodiment,lysis can be achieved using a chemical reagent, such as proteinase K,and a chaotropic salt solution. Said chemical reagents can be stored inone of more tubule segments and combined with the sample using theprocesses disclosed above. In some embodiments, multiple processes suchas chemical cell lysis, mechanical grinding and heating, can be combinedto break up solid sample, for example tissue collected from biopsy, tomaximize the performance.

Capturing target micro-organisms can be achieved by using a substrate.In an embodiment, the surface of the substrate may be coated with atleast one binding reagent, such as an antibody, ligand or receptoragainst an antigen, receptor or ligand on the surface of the targetorganism (ASA), a nucleic acid (NA), a peptide nucleic acid (PNA) andphosphothioate (PT) nucleic acid probe to capture a specific nucleicacid target sequence complementary to the probe or a target organism. Inanother embodiment, the surface may be selected to have, or coated toform, an electrostatically charged (EC) surface, such as silica- or ionexchange resin-coated surface, to reversibly capture substantially onlynucleic acids. In some embodiments, the substrate may be pre-packed in atubule segment or sub-segment in dry format, and a liquid binding buffermay be packed in another segment. The substrate and the buffer can bereconstituted by using the aforementioned processes.

In some embodiments, a reagent from an adjoining segment can be used todilute the sample before incubation with the substrate. In someembodiments, the target organisms can be captured to the substrate priorto lysing the microorganisms; while in other embodiments, a lysis stepcan be conducted before the target capturing step. In preferredembodiments, incubation of the substrate in agitation can be conductedat a desired temperature, for example, at 4° C. for live bacterialcapture, or room temperature for viral capture. Capture can be followedby a washing process to remove the residues and unwanted components ofthe sample from the tubule segment.

In some embodiments, magnetic beads can be used as the substrate forcapturing target, and a magnetic bead immobilization and re-suspensionprocess may be used to separate the beads from the sample liquid. Inother embodiments where the substrate may be a pad 30 or a sheet 214(FIGS. 5A-B), the substrate 30 and 214 may be incorporated into thecollection tool 36 and/or may be adhered on the tubule wall in asegment.

Elution can be achieved by heating and/or incubating the substrate in asolution in a tubule segment at an elevated temperature. Preferredtemperatures for elution are from 50° C. to 95° C. In anotherembodiment, elution may be achieved by changing the pH of the solutionin which the substrate is suspended or embedded. For example, in anexemplary embodiment the pH of the wash solution can be between 4 and5.5 while that of the elution buffer can be between 8 and 9.

A spore germination process can be conducted by mixing a samplecontaining bacterial spores with germination solution, and incubatingthe mixture at a suitable condition. The germinant solution may containat least one of L-alanine, inosine, L-phenylalanine, and/or L-proline aswell as some rich growth media to allow for partial growth of thepre-vegetative cells released from the spores. Preferred incubationtemperatures for germination range from 20° C. to 37° C. By coating thesubstrate with an anti-spore antibody, vegetative cells can beselectively enriched from a sample that contains both live and/or deadspores. The live spores can release a plurality of vegetative cells fromthe substrate, which can be further processed to detect nucleic acidsequences characteristic of the bacterial species. In some embodiments,the germinant solution can be absorbed in a pad.

In certain embodiments, nucleic acids extracted from the biologicalsamples may be further processed by amplifying the nucleic acids usingat least one method from the group: polymerase chain reaction (PCR),rolling circle amplification (RCA), ligase chain reaction (LCR),transcription mediated amplification (TMA), nucleic acid sequence basedamplification (NASBA), and strand displacement amplification reaction(SDAR). In some embodiments, the nucleic acids extracted from theorganism can be ribonucleic acids (RNA) and their processing may includea coupled reverse transcription and polymerase chain reaction (RT-PCR)using combinations of enzymes such as Tth polymerase and Taq polymeraseor reverse transcriptase and Taq polymerase. In some embodiments,nicked-circular nucleic acid probes can be circularized using T4 DNAligase or Ampligase™ and guide nucleic acids, such as DNA or RNAtargets, followed by detecting the formation of the closed circularizedprobes after an in vitro selection process. Such detection can bethrough PCR, TMA, RCA, LCR, NASBA or SDAR using enzymes known to thosefamiliar with the art. In exemplary embodiments, the amplification ofthe nucleic acids can be detected in real time by usingfluorescent-labeled nucleic acid probes or DNA intercalating dyes aswell as a photometer or charge-coupled device in the molecular analyzerto detect the increase in fluorescence during the nucleic acidamplification. These fluorescently-labeled probes use detection schemeswell known to those familiar in the art (i.e., TaqMan™, molecularbeacons™, fluorescence resonance energy transfer (FRET) probes,scorpion™ probes) and generally use fluorescence quenching as well asthe release of quenching or fluorescence energy transfer from onereporter to another to detect the synthesis or presence of specificnucleic acids.

A real-time detection of a signal from a tubule segment can be achievedby using a sensor 492 (FIG. 1B), such as a photometer, a spectrometer, aCCD, connected to a block, such as block 490. In exemplary embodiments,pressure can be applied by an actuator 392 on the tubule segment 190 tosuitably define the tubule segment's shape. The format of signal can bean intensity of a light at certain wavelength, such as a fluorescentlight, a spectrum, and/or an image, such as image of cells or manmadeelements such as quantum dots. For fluorescence detection, an excitationof light from the optical system can be used to illuminate a reaction,and emission light can be detected by the photometer. To detect aplurality of signals having specific wavelengths, different wavelengthsignals can be detected in series or parallel by dedicated detectionchannels or a spectrometer.

The disclosed devices and methods can be widely applied in the practiceof medicine, agriculture and environmental monitoring as well as manyother biological sample testing applications. Nucleic acids isolatedfrom tissue biopsy samples that surround tumors removed by a surgeon canbe used to detect pre-cancerous tissues. In these applications, hot-spotmutations in tumor suppressor genes and proto-oncogenes can be detectedusing genotyping techniques well known to those familiar with the art.Pre-cancerous tissues often have somatic mutations which can readily beidentified by comparing the outcome of the genotyping test with thebiopsy sample to the patient's genotype using whole blood as a source ofnucleic acids. Nucleic acids isolated from white blood can be used todetect genetic variants and germline mutations using genotypingtechniques well known to those familiar with the art. Examples of suchmutations are the approximately 25 known mutants of the CFTR generecommended for prenatal diagnosis by the American College of MedicalGenetics and the American College of Obstetricians and Gynecologists.Examples of genetic variants are high frequency alleles inglucose-6-phosphate dehydrogenase that influence sensitivity totherapeutic agents, like the antimalarial drug Primaquine.

Another example of genetic variations with clinical relevance arealleles pertaining to increased risks of pathological conditions, likethe Factor V Leiden allele and the increased risk of venous thrombosis.Nucleic acids isolated from bacteria can be used to detect gene codingsequences to evaluate the pathogenicity of a bacterial strain. Examplesof such genes are the Lethal Factor, the Protective Antigen A, and theEdema factor genes on the PXO1 plasmid of Bacillus anthracis and theCapsular antigen A, B, and C on the PXO2 plasmid of the B. anthracis.The presence of these sequences allows researchers to distinguishbetween B. anthracis and harmless soil bacteria. Nucleic acids isolatedfrom RNA viruses can be used to detect gene coding sequences to detectthe presence or absence of a virus or to quantify a virus in order toguide therapeutic treatment of infected individuals.

A particularly significant utility of such assays is the detection ofthe human immunodeficiency virus (HIV), to guide anti-retroviraltherapy. Nucleic acids isolated from DNA viruses can be used detect genecoding sequences to detect the presence or absence of a virus in bloodprior to their use in the manufacturing of blood derived products. Thedetection of hepatitis B virus in pools of blood samples is a well-knownexample of this utility to those familiar in the art. The presence ofverotoxin Escherichia coli in ground beef is a good example of thepotential agricultural uses of the apparatus. Detecting the Norwalkvirus on surfaces is an example of a public health environmentalmonitoring application.

EXAMPLES Example 1 Genomic DNA Isolation and Detection from Whole Blood

DNA isolation and DNA sequence detection can be accomplished in a tube 1(FIG. 1B), including a flexible tubule 10 having nine segments separatedby peelable seals and containing pre-packed reagents, and a cap 20,having a waste reservoir 22 housed therein. The first segment 110 of thetubule can receive the whole blood sample. The second segment maycontain dilution buffer having 40 μl of phosphate buffered saline (PBS)221 (which may contain 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.4 mMKH₂PO₄, pH 7.3) and 250 μg dry proteinase K 222, which can be housed insub-segment one 121 and two 122 respectively, separated by a peelableseal 125. The third segment 130 may contain 50 μl of lysis buffer 230that may contain chaotropic salts which may contain 4.7 M guanidiniumhydrochloride, 10 mM urea, 10 mM Tris HCl, pH 5.7, and 2% triton X-100.The fourth segment 140 may contain 500 μg of magnetic silica beads 240,such as MagPrep™ beads (Merck & Co), suspended in 80 μl of isopropanol.These beads can bind DNA in the presence of chaotropic salts andalcohol. The fifth segment 150 may contain 80 μl of wash buffer 250(which may contain 50% ethanol, 20 mM NaCl, 10 mM Tris HCl, pH7.5). Thesixth segment 160 may contain 80 μl of 20 mM 2-morpholinoethanesulfonicacid (MES) buffer 260, pH 5.3. The pH of the MES buffer may be adjustedsuch that it can be low enough to avoid DNA elution from the beads. Theseventh segment 170 may contain 80 μl elution buffer 270 (10 mM TrisHCl, pH 8.5: an example of a buffer suitable for PCR). The pH of theelution buffer may be adjusted such that it can be high enough to elutethe DNA from the surface of the beads into the buffer. The eighthsegment 180 may contain dry uracil-N-glycosylase (UNG) 280. The ninthsegment 190 may contain dried PCR reagents 290 (which may contain 10nmol of each one of the 3 deoxynucleotide triphosphates (dNTPs):deoxyadenosine triphosphate (dATP), deoxycytosine triphosphate (dCTP),and deoxyguninosine triphosphate (dGTP); 20 nmol deoxyuridinetriphosphate (dUTP), 2.5 μmol of KCl, 200 nmol of MgCl₂, 1-5 units ofTaq DNA polymerase, and 20-100 pmol of each of the oligonucleotideprimers, and 6-25 pmol of TaqMan probe). The end 194 of the segment 190,can be permanently sealed or contain a pressure gate for collecting theproducts of the amplification reaction to confirm the results of agenotyping test by DNA sequencing or some other test known to thoseskilled in the art.

For genotyping, over 10 μl of whole blood may be loaded into the firstsegment 110. The tubule can then be closed by a cap 20 and inserted intoan analyzer. Sample processing may include the following steps.

1. Sample Lysis. All clamps, except the first clamp 310, may be closedon the tubule. The first actuator 312 may compress the first segment 110to adjust the volume of blood 210 to retain about 10 μl in the segment,and then the first clamp 310 may compress the tubule to close thesegment. The second actuator 322 can then compress the second segment120 (subsegments 121 and 122) to break the peelable seal 125 and mix PBS221 with proteinase K 222. The second clamp 320 can then open, and thesecond actuator can compress the second segment to open the peelableseal. The first and second actuators may further alternately compressthe segments to mix the dilution buffer with the blood sample. Theanalyzer can close the first actuator 312 and second clamp 320 to movethe diluted sample to the second segment 120, and move the third clamp330 to open and actuator 322 and 332 to alternately compress the tubulesegments 130 and 120 to open the peelable seal in-between the segmentsto mix the lysis buffer 230 with the diluted sample, and incubate themixture at 50° C. for 5 minutes. The incubation temperature can bemaintained by contact between the tubule and the thermal elementsincorporated within the actuators and/or blocks opposing the actuators.

2. Nucleic Acid Capture. After incubation, the fourth clamp 340 can openand the fourth actuator 342 may compress the fourth segment 140 to openthe peelable seal and mix the magnetic silica beads suspended inisopropanol 240 with the lysate in segments 130 and/or 120. Theactuators 322 and 332 with an adjacent actuator 312 or 342 canalternately compress their respective segments to agitate and incubatethe mixture for 5 minutes at room temperature to facilitate DNA bindingto the magnetic silica beads. Then, a magnetic field can be generated bya magnetic source 430 near the segment 130 to capture the beads insuspension. The actuator 322 and 332 can alternately compress segment120 and 130 to capture beads. As an alternative, the actuator 332 cancompress segment 130 to faun a flow-channel, and two flanking actuators322 and 342 can compress their respective segments alternately toincrease the capture efficiency. Substantially all the beads can beimmobilized on the wall of segment 130, then the actuators and clampsfrom actuator 342 to clamp 310 can be sequentially opened and closed tomove the unbound sample and waste to the waste reservoir 22.

3. Wash. A wash process can follow the capture process in order toremove residual debris and reaction inhibitors from the beads and thesegments that would be used for further sample processing. In thisembodiment, a dilution based washing can be used with the ethanol washbuffer and a thin-layer flow based washing can be used with the MES washbuffer. Clamps 350 and actuator 342 can first open, and then actuator352 can close to move the ethanol buffer 250 to segment 240, followed bythe closing of clamp 350. By using the same process on segments 140 and130, the ethanol buffer can be moved td segment 130. The magnetic fieldcan be removed; the actuator 332 and at least one adjacent actuator canbe alternately compressed against their respective segments to generateflow to re-suspend the beads. The magnetic field can then be turned onto capture substantially all the beads and the liquid can be moved towaste reservoir by using the processes mentioned above. After completingthe first wash, the MES wash buffer can be moved from segment 160 to140. Actuator 332 and clamp 340 and 330 can be gently released to form athin-layer flow channel through segment 130. Actuator 342 can compressgently on segment 140 to generate a certain inner pressure to ensure asubstantially uniform gap of the thin-layer flow channel. Actuator 342can then gently compress the tubule, and actuator 322 can release thetubule to ensure an essentially laminar flow of the wash buffer throughthe flow channel. When the wash is completed, the actuators and clampscan close and substantially all the waste may be moved to the wastereservoir 22.

4. Nucleic Acid Elution. The elution buffer 270 may then be moved fromsegment 170 to 130 by using a similar process as mentioned before. Themagnetic field can be removed and the beads can be re-suspended in theelution buffer under flow between segments 130 and 140. The beadsuspension can be incubated at 95° C. under stationary, flow oragitation conditions for 2 minutes. The magnetic field may be turned onand substantially all the beads can be immobilized, and the elutednucleic acid solution can be moved to segment 170 by sequentiallyopening and closing the actuators and clamps. The actuator 372 cancompress segment 170 to adjust the volume of the eluted nucleic acidsolution to 50 μl and clamp 370 can then close against the tubule tocomplete the DNA extraction process.

5. Nucleic Acid Amplification and Detection. The nucleic acid solutioncan then be transferred to segment 180, mixed, and incubated with UNG280 at 37° C. for 5 minutes to degrade any contaminant PCR products thatmay have been present in the biological sample. After the incubation,the temperature may be increased to 95° C. to denature DNA and UNG for 2minutes. The nucleic acid solution can then be transferred to segment190, and mixed with PCR reagent 290 at 60° C. to initiate hot start PCR.A typical 2-temperature, amplification assay of 50 cycles of 95° C. for2 seconds and 60° C. for 15 seconds can be conducted by setting segment180 at 95° C. and segment 190 at 60° C., and transferring the reactionmixture between the segments alternately by closing and opening actuator382 and 392. A typical 3-temperature, amplification assay of 50 cyclesof 95° C. for 2 seconds, 60° C. for 10 seconds, and 72° C. for 10seconds can be conducted by setting segment 170 at 95° C., segment 180at 72° C. and segment 190 at 60° C., and alternately transferring thereaction mixture among the segments by closing and opening the actuators372, 382 and 392. A detection sensor 492, such as a photometer can bemounted on the block 394 to monitor real-time fluorescence emission fromthe reporter dye through a portion of the tubule wall. After an assay iscomplete, the test results can be reported and the sample can betransferred to segment 198 through the pressure gate 194 by compressingsegment 190 for further processing.

Ten microliters of fresh Ethylenediamine Tetraacetic Acid (EDTA)-treatedhuman whole blood were loaded into a pre-packed sample tube andprocessed on an analyzer as described in the text. Detection wasaccomplished with a VIC™-labeled TaqMan Minor Groove Binder probecomplimentary to the wild-type hemochromatosis (HFE) gene and aFAM-labeled TaqMan Minor Groove binder probe complementary to the C282Ymutant. FIG. 8 shows the results of three independent experiments, and anegative control in which template DNA was omitted. As these samplescontained only wild-type HFE alleles, only the VIC fluorescence trace isshown.

Example 2 Genomic DNA Isolation and Detection from Swab Sample

DNA isolation and DNA sequence detection can be performed in a tube 1,including a flexible tubule 10 having nine segments separated bypeelable seals and containing pre-packed reagents, and a cap 20, havinga waste reservoir 22 housed therein and additionally a swab protrudingfrom the cap opening. All pre-packed reagents may be identical to thatin Example 1, except that sub-segment one 121 of the second segment 120may contain 50 μl PBS dilution buffer.

The swab on cap 20 can be used to collect a sample from the oral cavity,a surface, or other swabable samples known to those skilled in the art.After collection, the cap can be mated to the tubule, introducing theswab sample to the first segment 110. The tubule can then be insertedinto an analyzer. All clamps, except the first clamp 310, may be closedon the tubule. The second actuator 322 can compress the second segment120 (subsegments 121 and 122) to break the peelable seal 125 and mix PBS221 with proteinase K 222. The second clamp 320 can then open, and thesecond actuator compress the second segment to open the peelable sealand move the PBS and proteinase K reagents into the first segment 110.The clamp 320 can close and the first actuator 312 alternately compressand releases to elute the swab sample from the swab tip. After thesample is eluted, the first actuator 312 can compress the first segment110 and the clamp 320 and second actuator 322 can open to allow thetransfer of the eluted sample into the second segment. The secondactuator 322 can then compress on the second segment 120 to adjust thevolume of eluted sample to about 50 μl, and the second clamp 320 canthen compress the tubule to close the segment. All subsequent sampleprocessing steps are similar to that described in Example 1.

A rayon-tipped sterile swab (Copan, Italy) was scraped against theinside of donor's cheek to harvest buccal epithelial cells. Swab wasdipped into 20 μl PBS and stirred briskly to suspend cells. Tenmicroliters of suspended cells were loaded into a pre-packed sampletubule and processed in an analyzer as described in the text. Detectionwas accomplished with a VIC-labeled TaqMan Minor Groove Binder probecomplimentary to the wild-type HFE gene, and a FAM-labeled probecomplimentary to the 282Y mutant of the HFE gene (FIG. 9).

Example 3 Bacterial DNA Isolation from Plasma

DNA isolation and DNA sequence detection from plasma can be performed ina tube 1, including a flexible tubule 10 having nine segments separatedby peelable seals and containing pre-packed reagents, and a cap 20,having a waste reservoir 22 housed therein. All pre-packed reagents canbe identical to that in example 1, except that sub-segment one 121 ofthe second segment 120 can contain 50 μl PBS dilution buffer, the thirdsegment 130 can contain 100 μl of lysis buffer 230, and the fourthsegment 140 can contain 500 μg of silica magnetic beads suspended in 130μl of isopropanol. For bacterial DNA detection, over 10 μl of plasma maybe loaded into the first segment 110. The sample can then be processedusing the pre-packed reagents with the sample processing steps describedin Example 1.

Approximately 10⁵ E. coli O157:H7 cells were diluted to a volume of 10μl in human plasma used for the assay. DNA extraction and detection wereperformed in the analyzer as described. A FAM-labeled probe recognizingthe Stx1 gene of O157:H7 was used for detection. FIG. 10 shows theresults with a negative control in which E. coli O157:H7 DNA wasomitted.

Example 4 Viral RNA Isolation and Detection from Plasma

RNA isolation and RNA sequence detection from plasma can be performed ina tube 1, including a flexible tubule 10 having nine segments separatedby peelable seals and containing pre-packed reagents, and a cap 20,having a waste reservoir 22 housed therein. All pre-packed reagents canbe identical to that in Example 3, except that the fourth segment 140can contain either a silica membrane, silica sheet, or silica fiber meshsized to fit entirely within the segment, as well as 130 μl ofisopropanol; and the ninth segment 190 can contain dried RT-PCR reagents290 which can include 10 nmol of each one of; dATP, dCTP, and dGTP; 20nmol dUTP, 2.5 μmol of KCl, 200 nmol of MgCl₂, 1-5 units of Tth DNApolymerase, and 20-100 pmol of each of the oligonucleotides primer, and6-25 pmol of TaqMan probe, with or without 1-5 units of Taq DNApolymerase.

For viral nucleic acid isolation and detection, over 50 μl of plasma canbe loaded into the first segment 110. The sample can then be processedusing the pre-packed reagents with the sample processing steps describedin Example 1, with the exception of a modified nucleic acid capture stepand an additional reverse transcription step. For the nucleic acidcapture step, the fourth clamp 340 may open and the fourth actuator 342may compress the fourth segment 140 to open the peelable seal and allowthe lysate 230 to come into contact with the silica membrane inisopropanol 240 in segment 130. The actuators 332 and 342 canalternately compress their respective segments to agitate and incubatethe mixture for 5 minutes, at room temperature to facilitate nucleicacid binding to the silica membrane. Following nucleic acid capture, theactuator 342 can compress the segment 140 and the liquid waste can bemoved to the waste reservoir. The clamp 330 can close and actuators 332,342, and 352 can form a flow channel in segments 130, 140, and 150 toallow the ethanol wash buffer to wash the substrate. All subsequentsample processing steps can be the same as Example 3. The additionalreverse transcription step may occur prior to PCR amplification andincludes incubation of the extracted RNA with RT-PCR reagents in theninth segment 190 at 65° C. for 10 minutes.

Example 5 Bacterial DNA Isolation and Detection from Whole Blood

DNA isolation and DNA sequence detection from whole blood can beperformed in a tube 1, including a flexible tubule 10 having ninesegments separated by peelable seals and containing pre-packed reagents,and a cap 20, having a waste reservoir 22 housed therein. Sub-segmentone 121 of the second segment 120 may contain 50 μl PBS dilution buffer,the third segment 130 may contain 100 μl of lysis buffer 230, and thefourth segment 140 may contain 10 μg of magnetic beads such asDynabeads™ (Dynal Biotech), conjugated to 10⁴ to 10⁷ copies of a peptidenucleic acid (PNA) probe, suspended in hybridization buffer (100 μl of2×SSC/0.1 M EDTA). All other pre-packed reagents can be the same as thatdescribed in Example 1.

For bacteria nucleic acid isolation and detection, over 50 μl of wholeblood can be loaded into the first segment 110. The sample can then beprocessed using the pre-packed reagents with the sample processing stepsdescribed in Example 1, with the exception of a modified nucleic acidcapture step. For the nucleic acid capture step, the fourth clamp 340opens and the fourth actuator may compress the fourth segment 140 toopen the peelable seal and mix the PNA-coupled magnetic beads suspendedin hybridization buffer 240 with the lysate in segment 130. Theactuators 322 and 332 with an adjacent actuator 312 or 342 mayalternately compress their respective segments to agitate and incubatethe mixture for 15 minutes at room temperature to facilitate DNAhybridization to the PNA probes coupled to magnetic beads. The samplecan then be processed using the pre-packed reagents with the sampleprocessing steps described in Example 1.

Example 6 Viral RNA Isolation and Detection from Whole Blood

Viral RNA isolation and RNA sequence detection from plasma can beperformed in a tube 1, including a flexible tubule 10 having ninesegments separated by peelable seals and containing pre-packed reagents,and a cap 20, having a waste reservoir 22 housed therein. All pre-packedreagents may be identical to that in Example 5, except that the ninthsegment 190 may contain dried RT-PCR reagents 290 which may include 10nmol of each one of dATP, dCTP, and dGTP; 20 nmol dUTP, 2.5 μmol of KCl,200 nmol of MgCl₂, 1-5 units of Taq DNA polymerase, 1-5 units of Tth DNApolymerase, and 20-100 pmol of each of the oligonucleotide primers, and6-25 pmol of TaqMan probe. For viral RNA isolation and detection, over50 μl of whole blood is loaded into the first segment 110. The samplecan then be processed using the pre-packed reagents with the sampleprocessing steps described in Example 1, with the exception of anadditional reverse transcription step, prior to amplification, in whichthe extracted RNA is incubated with RT-PCR reagents in the ninth segment190 at 65° C. for 10 minutes.

Example 7 Bacterial Isolation Using Immunomagnetic Enrichment from WholeBlood

Bacterial DNA isolation and DNA sequence detection from whole blood canbe performed in a tube 1, including a flexible tubule 10 having ninesegments separated by peelable seals and containing pre-packed reagents,and a cap 20, having a waste reservoir 22 housed therein. The secondsegment 120 may contain dry magnetic beads, such as Dynabeads, coatedwith a capture antibody specific for a bacterial epitope. The thirdsegment 130 may contain 100 μl of PBS buffer 230 used to control thesample pH and dilute the red blood cell concentration to ensureefficient binding by the capture antibody. The fourth segment 140 maycontain red blood cell lysis buffer including dry salts (1 μmol KHCO₃,15 μmol NH₄Cl) and 100 μl of 0.1 mM EDTA, pH 8.0 buffer housed in twosub-segments separated by peelable seal. The fifth segment 150 and sixthsegment 160 may contain 80 μl of PBS wash buffer, respectively. Allother pre-packed reagents are identical to that in Example 1.

For bacterial detection in whole blood, over 50 μl of whole blood can beloaded into the first segment 110. The tubule is then closed by a cap 20and inserted into an analyzer. Sample processing includes the followingsteps.

1. Target Cell Capture. All clamps, except the first clamp 310, may beclosed on the tubule. The first actuator 312 may compress on the firstsegment 110 to adjust the volume of blood 210 to about 50 μl remainingin the segment, and then the first clamp 310 may compress the tubule toclose the segment. The third actuator 332 can then compress the thirdsegment 130 to break the peelable seal between segment 130 and segment120 to mix PBS buffer with antibody coupled magnetic beads toreconstitute a capture solution. The second clamp 320 can then open, andthe first actuator 312 can compress the segment 110 to move the bloodsample to the second segment 120 and third segment 130. The secondactuators 322 and third actuator 332 can then alternately compress thesegments to mix the capture solution with blood sample while incubatingthe mixture at 4° C. for 15-30 minutes to facilitate antibody binding tothe target cells. Then, a magnetic field generated by a magnetic source430 can be applied on the segment 130 to capture the beads insuspension. The actuator 322 and 332 can alternately compress segment120 and 130 to capture beads. After substantially all the beads areimmobilized on the wall of segment 130, the actuators and clamps fromactuator 332 to clamp 310 can sequentially open and close to move theunbound sample and waste to the waste reservoir 22.

2. Red Blood Cell Lysis. After target capture, the fourth clamp 340opens and the fourth actuator can compress the fourth segment 140 toreconstitute the red blood cell lysis buffer and move the buffer to thesegment 230. The magnetic field generated by a magnetic source 430 canbe removed to allow bead re-suspension. The actuator 322 and 332 canalternately compress their respective segments to agitate and incubatethe mixture for 5 minutes at room temperature to facilitate the lysis ofred blood cells remaining in the sample. Then, the magnetic field can beapplied to the segment 130 to capture the beads in suspension. Aftersubstantially all the beads are immobilized on the wall of segment 130,the unbound sample and waste can be moved to the waste reservoir 22.

3. Wash. Two wash processes can follow the binding step, both may usePBS wash buffer pre-packed in segments 150 and 160. Wash may occur bydilution-based wash using the process described above.

4. Nucleic Acid Elution. Elution can occur by the process described inExample 1. The beads suspension can be incubated at 95° C. understationary, flow or agitation conditions for 2-5 minutes to lyse thecaptured target cells and release DNA.

5. Nucleic Acid Amplification and Detection. Real-time PCR detection mayoccur by the same process as that described in Example 1.

Example 8 Viral RNA Isolation Using Immunomagnetic Enrichment from WholeBlood

Viral RNA isolation and sequence detection from whole blood can beperformed in a tube 1, including a flexible tubule 10 having ninesegments separated by peelable seals and containing pre-packed reagents,and a cap 20, having a waste reservoir 22 housed therein. All pre-packedreagents can be identical to those in Example 5, except that the secondsegment 120 may contain dry magnetic beads, such as Dynabeads, coatedwith a capture antibody specific for a viral epitope, and the ninthsegment 190 may contain dried RT-PCR reagents 290 which may include 10nmol of each one of dATP, dCTP, and dGTP; 20 nmol dUTP, 2.5 μmol of KCl,200 nmol of MgCl₂, 1-5 units of Taq DNA polymerase, 1-5 units of Tth DNApolymerase, and 20-100 pmol of each of the oligonucleotide primers, and6-25 pmol of TaqMan probe. For viral RNA isolation and sequencedetection, over 50 μl of whole blood can be loaded into the firstsegment 110. The sample can then be processed using the pre-packedreagents with the sample processing steps described in Example 7, withthe exception of a modified target capture step and an additionalreverse transcription step. For the target capture step, virion captureby antibody-coupled magnetic beads can be performed at room temperaturefor 5 minutes in segments 120 and 130. The reverse transcription stepmay occur prior to amplification, and includes incubation of theextracted RNA is with RT-PCR reagents in the ninth segment 190 at 65° C.for 10 minutes.

Example 9 Multiplex Genotyping of Human DNA with Padlock Probes andMelting Curve Analysis

DNA isolation and DNA sequence detection from whole blood may beperformed in a tube 1, including a flexible tubule 10 having ninesegments separated by peelable seals and containing pre-packed reagents,and a cap 20, having a waste reservoir 22 housed therein. All pre-packedreagents may be identical to those listed in Example 1, with theexception of the eighth segment 180 and the ninth segment 190. Theeighth segment 180 may include two sub-segments separated by peelableseal; the first sub-segment may contain dry padlock probes and T4 DNAligase 280, and the second sub-segment may contain dry exonucelase I andexonucelase III. The ninth segment 190 may contain dry UNG and PCRreagents 290 (which can include 200 μmol of each one of the 3 dNTPs, 100pmol of each of the oligonucleotides used by PCR, 400 μmol dUTP, 1 nmolof KCl, 0.1 nmol of MgCl₂, 5 units of Taq DNA polymerase and optionally12.5 pmol of TaqMan probe or molecular beacon).

For genotyping, over 10 μl of whole can be loaded into the first segment110. The sample can then be processed using the pre-packed reagents withthe sample processing steps described in Example 1, with the exceptionof the nucleic acid amplification and detection step. After nucleic acidextraction is complete in the seventh segment 170, actuator 372 mayadjust the volume of nucleic acid solution in segment 170 toapproximately 5-15 μl, while the remainder of the nucleic acid solutionis held in segment 160, segregated from segment 170 by clamp 370. Theactuator 372 may then compress on segment 170 to burst the peelable sealbetween the segment 170 and 180, while maintaining the peelable sealbetween the first and second sub-segments of segment 180. The extractednucleic acids may be mixed with T4 DNA ligase and padlock probes in thefirst sub-segment of segment 180, and the mixture may be moved tosegment 170. The remaining nucleic acid solution held in segment 160 mayalso be moved to segment 170. The nucleic acid solution, padlock probeand T4 ligase may be incubated in segment 170 at 37° C. for 15 minutes.The mixture may then be moved to the eighth segment 180 to break thepeelable seal of the second sub-segment of segment 180 to incubate thenucleic acids with Exonuclease I and Exonuclease III at 37° C. for 5minutes to degrade all linear DNA fragments. After incubation, thesolution may be heated to 95° C. in the eighth segment 180 to inactivatethe Exonucelases and T4 ligase. The solution can then be transferred tothe ninth segment 190 to mix with dry UNG and PCR reagents. The UNGdegrades any contaminant PCR products that may have been present whenthe sample was introduced, and linearizes the circularized padlockprobes to facilitate the amplification of the reporter sequences. PCRamplification may be performed as described in Example 1. A detectionsensor 492 mounted on the block 394 can monitor real-time fluorescenceemission from the reporter dye through a portion of the tubule wall.Melting curve analysis can be performed to identify the targets.Alternatively, the sample can be transferred to segment 198 through thepressure gate 194 for further detection on a nucleic acid microarray orother detection techniques known to those skilled in the art.

Example 10 Live Bacterial Spore Isolation and Germination

DNA isolation and DNA sequence detection from surface swab spore samplecan be performed in a tube 1, including a flexible tubule 10 having ninesegments separated by peelable seals and containing pre-packed reagents,and a cap 20, having a waste reservoir 22 housed therein andadditionally a swab protruding from the cap opening. The first segment110 of the tubule may include two sub-segments separated by a peelableseal; the first sub-segment can be adapted to housing a swab sample, andthe second sub-segment may contain 80 μl of PBS wash buffer having a pHappropriate to permit efficient binding of the spores by the captureantibody. The second segment 120 may contain solid substrate whereonanti-spore antibodies may be coated; wherein the antibodies have a highaffinity for epitopes on the spore and low affinity for epitopes on thegerminated cell. The second segment may be further pre-packed with avolume of a gas to facilitate breaking of the peelable seal betweensegments 120 and 110. The third segment 130 may contain 50 μl, of sporegermination reagents 230 which may include Brain Heart infusion medium(Difco), His 50 mM, Tyr 1 mM, Inosine 2 mM, Ala 200 mM, and Ser 200 mM.The fourth segment 140 may contain 50 μl of lysis buffer 240 containingchaotropic salts including 4.7 M guanidinium hydrochloride, 10 mM urea,10 mM Tris HCl, pH 5.7, and 2% triton X-100. The fifth segment 150 maycontain 500 μg of magnetic silica beads 240, such as MagPrep™ beads(Merck & Co), suspended in 80 μl of isopropanol. The sixth segment 160may contain 80 μl of wash buffer (50% ethanol 250, 20 mM NaCl, 10 mMTris HCl, pH 7.5). The seventh segment 170 may contain 80 μl of 20 mMMES buffer 270, pH 5.3. The eighth segment 180 may contain 80 μl elutionbuffer 280 (10 mM Tris HCl, pH 8.5). The ninth segment 190 may containdry UNG and dried PCR reagents 290 (which may include 10 nmol of eachone of the dATP, dCTP, and dGTP; 20 nmol dUTP, 2.5 μmol of KCl, 200 nmolof MgCl₂, 1-5 units of Taq DNA polymerase, and 20-100 pmol of each ofthe oligonucleotide primers, and 6-25 pmol of TaqMan probe).

For live spore detection, the swab integrated into the cap 20 can beused to collect a sample. After collection, the cap can be mated to thetubule, introducing the swab sample to the first segment 110. The tubulecan then be inserted into an analyzer. Sample processing may include thefollowing steps.

1. Spore germination. All clamps, except the first clamp 310, may beclosed on the tubule. The first actuator 312 compresses on the firstsegment 110 to burst the peelable seal between the first and secondsub-segment of segment 110 to release the PBS wash buffer. The firstactuator 310 may then alternately compress and decompress the segment110 to wash spores from the swab head using the PBS buffer. Aftersuspension of the spores in PBS, actuator 322 may compress segment 120to burst the peelable seal between segments 110 and 120 and allow thespore suspension to move to segment 120. Clamp 320 can close andactuator 322 can alternately compress segment 120 to facilitate bindingof the spore to the antibody. After incubation, the liquid waste can bemoved to the waste reservoir. Actuator 332 can then compress segment 130to burst the peelable seal between segments 120 and 130 to allow thegermination solution to be incubated with the captured spores at 37° C.for 13 minutes with agitation in segment 120. Germinated cells will notbe bound by the spore-specific antibody and will be suspended insolution.

2. Nucleic Acid Capture. After germination, the fourth clamp 340 canopen and the fourth actuator 342 compress the fourth segment 140 to openthe peelable seal and mix the lysis buffer with the germinated cells.Then the fifth clamp 350 can open and the fifth actuator 352 compresssegment 150 to move magnetic silica beads suspended in isopropanol 240to segment 130 to mix with the lysate. The actuators 332 and 342 canalternately compress their respective segments to agitate and incubatethe mixture for 5 minutes at room temperature to facilitate DNA bindingto the magnetic silica beads. Then, the magnetic field generated by amagnetic source 430 can be applied on the segment 130 to capture thebeads in suspension. The actuator 332 and 342 can alternately compresssegment 130 and 140 to capture beads. After substantially all the beadsare immobilized on the wall of segment 130, the unbound sample and wastecan be moved to the waste reservoir 22.

3. Wash. Ethanol wash buffer in segment 160 and MES buffer in segment170 can be used for washing the immobilized beads. A dilution based washcan be performed in segments 120 and 130 by actuators 322 and 332 asdescribed in Example 1. Alternatively, a thin-layer flow based wash canbe performed in segments 120, 130, and 140 by actuators 322, 332, and342 as described in Example 1.

4. Nucleic acid elution. Elution buffer 270 can be moved from segment180 to 130 for DNA elution as described in Example 1.

5. Nucleic Acid Amplification and Detection. The nucleic acid solutioncan then be transferred to segment 190 and mixed with UNG and dry PCRreagents. Incubation of the reaction mixture at 37° C. for 5 minutesallows UNG to degrade any contaminant PCR products. After theincubation, the reaction mixture can be transferred to segment 180 fordenaturation at 95° C. for 2 minutes. The nucleic acid solution can thenbe transferred to segment 190, for incubation at 60° C. to initiate hotstart PCR. A typical 2-temperature, amplification assay of 50 cycles of95° C. for 2 seconds and 60° C. for 15 seconds can be conducted bysetting segment 180 at 95° C. and segment 190 at 60° C., andtransferring the reaction mixture between the segments alternately byclosing and opening actuator 382 and 392. A typical 3-temperature,amplification assay of 50 cycles of 95° C. for 2 seconds, 60° C. for 10seconds, and 72° C. for 10 seconds can be conducted by setting segment170 at 95° C., segment 180 at 72° C. and segment 190 at 60° C., andalternately transferring the reaction mixture among the segments byclosing and opening the actuators 372, 382 and 392. A detection sensor492, such as a photometer can be mounted on the block 394 to monitorreal-time fluorescence emission from the reporter dye through the tubulewall. After an assay is complete, the test results can be reported andthe sample can be transferred to segment 198 through the pressure gate194 by compressing segment 190 for further processing.

Example 11 Multiplex Genotyping of Human DNA from Solid Tissue Sample

In a eleventh embodiment, DNA isolation and DNA sequence detection fromsolid tissue sample can be performed in a tube 1, including a flexibletubule 10 having nine segments separated by peelable seals andcontaining pre-packed reagents, and a cap 20, having a waste reservoir22 housed therein. The first segment 110 of the tubule can be adapted toreceive a solid tissue sample and have tough walls with micro-teeth-likeinner surfaces to facilitate tissue grinding. The second segment 120 cancontain 250 μg dry proteinase K 222. The third segment 130 can contain100 μl of lysis buffer 230 containing chaotropic salts including 4.7 Mguanidinium hydrochloride, 10 mM urea, 10 mM Tris HCl, pH 5.7, and 2%triton X-100. The fourth 140, fifth 150, sixth 160 and the seventh 170segments can contain the same reagents as in Example 1. The eighthsegment 180 can include two sub-segments separated by a peelable seal;the first sub-segment may contain dry padlock probes and T4 DNA ligase280, and the second sub-segment may contain dry exonuclease I andexonuclease III. The ninth segment 190 may contain dry UNG and PCRreagents 290 (which may include 200 μmol of each one of the 3 dNTPs, 100pmol of each of the oligonucleotides used by PCR, 400 μmol dUTP, 1 nmolof KCl, 0.1 nmol of MgCl₂, 5 units of Taq DNA polymerase and optionally12.5 pmol of TaqMan probe).

For a mutation detection assay, a 1 mg to 50 mg solid tissue sample canbe loaded into the first segment. The tubule can then be closed by a cap20 and inserted into an analyzer. Subsequently, all clamps can be closedon the tubule. The clamp 330 can open and the third actuator 332compress the third segment 130 to break the peelable seal betweensegment 120 and 130 to mix the lysis buffer 230 with proteinase K. Thesecond clamp 320 can then open, and the second actuator can compress thesecond segment to open the peelable seal and introduce the lysissolution to the solid tissue sample in segment 110. The second clamp 320can close, and the first actuator 312 can compress and decompress thesegment 110, facilitating the homogenization of the solid tissue samplewith the micro-teeth on the tubule wall surface. The thermal elementcontacting segment 110 may be set to 50-68° C. to increase theefficiency of proteinase digestion. After the tissue sample has beensufficiently homogenized, the homogenate can be moved to segment 120 andthe magnetic silica beads suspended in isopropanol of segment 140 can bemoved to segment 130. The actuators 322 and 332 can alternately compresstheir respective segments to mix the homogenate with the bead suspensionto facilitate DNA binding to the magnetic silica beads. Then, themagnetic field generated by a magnetic source 430 can be applied to thesegment 130 to capture the beads in suspension. The actuators 322 and332 can alternately compress segments 120 and 130 to capture beads inthe magnetic field. As an alternative, the actuator 332 can compresssegment 130 to form a flow-channel, and two flanking actuators 322 and342 can compress the respective segments alternately to increase thecapture efficiency. After substantially all the beads have beenimmobilized on the wall of segment 130, the actuators and clamps fromactuator 342 to clamp 310 can be sequentially opened and closed to movethe unbound sample and waste to the waste reservoir 22. The subsequentwash and nucleic acid elution steps can occur by the process describedin Example 1. Nucleic acid amplification and detection can occur by thepadlock probe assay process as described in Example 9.

Example 12 Plasma Separation and Virus Detection from Whole Blood

In a twelfth embodiment, RNA isolation and sequence detection from wholeblood can be performed in a tube 1, including a flexible tubule 10having nine segments separated by peelable seals and containingpre-packed reagents, and a cap 20, having a waste reservoir 22 housedtherein. The first segment 110 of the tubule can include twosub-segments separated by a peelable seal; the first sub-segment can beadapted to receive a whole blood sample, and second sub-segment cancontain one of a coagulant, such as thrombin, or a dry multi-valentanti-red blood cell antibody. The first segment further can contain atits base in the second sub-segment one or a plurality of embedded filterbags of pore size preferably between 1 μm to 10 μm. Filter pore size canbe such that substantially no blood cells may pass and only plasma maypass. The second segment 120 may contain 80 μl PBS dilution buffer. Thethird segment 130 may contain 250 μg dry proteinase K and 60 μl lysisbuffer (4.7 M guanidinium hydrochloride, 10 mM urea, 10 mM Tris HCl, pH5.7, and 2% triton X-100) housed in two sub-segments separated by apeelable seal. The fourth 140, fifth 150, sixth 160, seventh 170, andeighth 180 segments may contain the same reagents as in Example 1. Theninth segment 190 may contain dried RT-PCR reagents 290 which caninclude 10 nmol of each one of: dATP, dCTP, and dGTP; 20 nmol dUTP, 2.5μmol of KCl, 200 nmol of MgCl₂, 1-5 units of Taq DNA polymerase, 1-5units of Tth DNA polymerase, and 20-100 pmol of each of theoligonucleotides primer, and 6-25 pmol of TaqMan probe.

For plasma separation within the tubule, approximately 300 μl of wholeblood can be loaded into the first segment 110. All clamps can beclosed, and actuator 312 can compress segment 110 to burst the peelableseal between the sub-segments and allow the mixing of the blood samplewith dry multi-valent anti-red blood cell antibody or coagulant.Actuator 312 can alternately compress and decompression the segment 110to facilitate the binding of antibody to red blood cells and theformation of cell clusters. Actuator 322 can compress segment 120 toburst the peelable seal between segment 120 and 110 and to move thedilution buffer to segment 110 to mix with blood sample. After asufficient quantity of red blood cells have aggregated, actuator 312 cangently compress segment 110 to drive the blood sample through theembedded filter, while actuator 322 can slowly decompress segment 120 tocreate suction from the other side of the filter. Following plasmaseparation, clamp 320 can be closed and actuator 332 can compresssegment 130 to reconstitute dry proteinase K in the lysis buffer. Clamp330 can then open and actuator 322 can compress segment 120 to mix theplasma sample with the lysis buffer and incubate the mixture at 50° C.for 5 minutes in segment 130. For DNA viruses, the subsequent nucleicacid capture, wash, elution, and amplification and detection steps canbe the same as that described in Example 1. A reverse transcription stepmay be added prior to amplification, in which the extracted RNA isincubated with RT-PCR reagents in the ninth segment 190 at 65° C. for 10minutes.

Example 13 Genomic DNA Isolation and Detection from Whole BloodCollected on Cotton Based Matrices

In a thirteenth embodiment, DNA isolation and DNA sequence detection canbe accomplished in a tube 1, including a flexible tubule 10 having foursegments separated by peelable seals and containing pre-packed reagents,and a cap 20, which may have a waste reservoir 22 housed therein. Thefirst segment 110 of the tubule can receive the whole blood samplecollected on cotton-based matrices, such as Whatman BFC 180 and FTA®paper, Schleicher and Schuell 903™ and IsoCode® paper. The secondsegment 120 may contain washing buffer including 40 μl of distilledwater 220. The third segment 130 may contain 80 μl elution buffer (10 mMTris HCl, pH 8.5) or distilled water 230. The fourth segment 140 maycontain dry UNG and dried PCR reagents 240 (which may contain 10 nmol ofeach one of the 3 dNTPs: dATP, dCTP, and dGTP; 20 nmol dUTP, 2.5 μmol ofKCl, 200 nmol of MgCl₂, 1-5 units of Taq DNA polymerase, and 20-100 pmolof each of the oligonucleotide primers, and 6-25 pmol of TaqMan probe).The end of segment 140 can be permanently sealed.

For genotyping, whole blood, such as collected by a finger prick orother means may be absorbed onto cotton-based matrices 30 attached tosample tubule cap 20 through connector 36. The tube can then be closedby a cap 20 and inserted into an analyzer. Sample processing may includethe following steps.

1. Sample Lysis. All clamps, except the first clamp 310, may be closedon the tubule. The first actuator 312 may compress the first segment 110to adjust the distance of the actuator 312 to the cotton-based matrices30 in the segment, and then the first clamp 310 may compress the tubuleto close the segment. The first segment can be incubated at 95° C. for 5minutes to dry the blood sample. Then, the segment temperature may beallowed to cool to room temperature. The drying process can lyse wholeblood cells and enhance the binding of plasma proteins and PCRinhibitors to the cotton matrices. The incubation temperature can bemaintained by contact between the tubule and the thermal elementsincorporated within the actuators and/or blocks opposing the actuators.

2. Wash. A wash process can follow the heating process in order toremove washable residuals and PCR inhibitors from the matrices and thesegments that would be used for further sample process. In thisembodiment, a dilution based washing or a thin-layer flow based washingcan be used. For dilution based wash, Clamps 320 can first open, andthen actuator 322 can close to move the wash buffer 220 to segment 210,followed by the closing of clamp 320. The first actuator 312 can agitatethe cotton-based matrices through a repeated compressing and releasingaction to release unbound plasma protein components and PCR inhibitorfor 3 minutes at room temperature. After completing the wash, the washbuffer can be moved from segment 110 to waste reservoir 22 housed in thecap 20. Actuator 312, clamps 310 and 320 can be gently released to forma thin-layer flow channel through segment 110. Actuator 322 can compressgently on segment 120 to generate a certain inner pressure to ensure asubstantially uniform gap of the thin-layer flow channel. Actuator 322can then compress the tubule to generate essentially laminar flow of thewash buffer through the flow channel. When the wash is completed, theactuators and clamps can compress on the segments and substantially allthe waste may be moved to the waste reservoir 22.

3. Nucleic Acid Elution. The elution buffer 230 may then be moved fromsegment 130 to 110 by using a similar process as mentioned before. Thecotton-based matrix can be incubated at 95° C. under stationary, flow oragitation conditions for 2 minutes. The eluate can then be moved tosegment 130. The actuator 332 can compress segment 130 to adjust thevolume of the eluted nucleic acid solution to 50 μl and clamp 330 canthen close against the tubule to complete the DNA extraction process.

4. Nucleic Acid Amplification and Detection. The nucleic acid solutioncan then be transferred to segment 140, mixed, and incubated with UNGand PCR reagent 240 at 37° C. for 5 minutes to degrade any contaminantPCR products that may have been present when the sample was introduced.After the incubation, the temperature may be increased to 95° C. todenature DNA for 2 minutes followed by PCR reaction. A typical2-temperature, amplification assay of 50 cycles of 95° C. for 2 secondsand 60° C. for 9-15 seconds can be conducted by setting segment 180 at95° C. and segment 190 at 60° C., and transferring the reaction mixturebetween the segments alternately by closing and opening actuator 332 and342. A typical 3-temperature, amplification assay of 50 cycles of 95° C.for 2 seconds, 60° C. for 8-10 seconds, and 72° C. for 8-12 seconds canbe conducted by setting segment 120 at 95° C., segment 130 at 72° C. andsegment 140 at 60° C., and alternately transferring the reaction mixtureamong the segments by closing and opening the actuators 322, 332 and342. A detection sensor, such as a photometer 492, can be mounted on theblock 344 to monitor real-time fluorescence emission from the reporterdye through the tubule wall.

All of the patents and publications cited herein are hereby incorporatedby reference.

We claim:
 1. A sample processing tubule, comprising: at least threesegments, each of which is: defined by the tubule; fluidly isolated, atleast in part by a breakable seal; so expandable as to receive a volumeof fluid expelled from another segment; and so compressible as tocontain substantially no fluid when so compressed; wherein at leastthree segments each contains at least one reagent.
 2. The tubule ofclaim 1, wherein at least a portion of the tubule is transparent.
 3. Thetubule of claim 1, further comprising at least one pressure gate influid communication with at least one segment.
 4. The tubule of claim 1,further comprising at least one filter in the tubule.
 5. The tubule ofclaim 1, wherein at least one of the reagents includes a substancecapable of specific binding to a preselected component of a sample whenthe sample is added to the tubule.
 6. The tubule of claim 5, wherein thesubstance is coupled to a solid substrate.
 7. The tubule of claim 6,wherein the substance fauns a coating on the solid substrate.
 8. Thetubule of claim 6, wherein the solid substrate comprises at least one ofbeads, a pad, a filter, a sheet, an electrostatic surface, and a portionof a tubule wall surface.
 9. The tubule of claim 6, wherein thesubstrate comprises at least one of silica beads, magnetic beads, silicamagnetic beads, glass beads, nitrocellulose colloid beads, andmagnetized nitrocellulose colloid beads.
 10. The tubule of claim 6,wherein the substance comprises silica, and the substrate comprises afilter or a sheet.
 11. The tubule of claim 6, wherein the substratecomprises a pad formed at least in part from an absorbent materialcomprising at least one of paper, film, filter, foam, mesh, and fibermatrix.
 12. The tubule of claim 6, wherein the substrate is coupled to atubule wall.
 13. The tubule of claim 1, further comprising a substrate,wherein the substrate comprises a pad formed at least in part from anabsorbent material comprising at least one of paper, film, filter, foam,mesh, and fiber matrix.
 14. The tubule of claim 1, further comprising anopen end for introducing a sample into the tubule.
 15. The tubule ofclaim 14, further comprising a cap for closing the open end.
 16. Thetubule of claim 1, further comprising a frame to which the tubule ismounted.
 17. The tubule of claim 1, further comprising a proximal endhaving an opening through which a sample is introducible, and a distalend, and wherein a second segment is distal to a first segment, and athird segment is distal to the second segment.
 18. The tubule of claim1, wherein the segments form a substantially linear array.
 19. Thetubule of claim 1, wherein the segments form a contiguous array.
 20. Amethod of processing a sample, comprising: introducing a sample into atubule discretized by breakable seals into a plurality of fluidlyisolated segments, wherein the tubule has a proximal end for receivingwaste and a distal end for conducting an assay; incubating the sample ina segment of the tubule with a substance capable of specific binding toa preselected component of the sample; removing waste from thepreselected component by clamping the tubule distally of the segmentcontaining the preselected component and compressing that segment; andreleasing a reagent to mix with the preselected component from a fluidlyisolated adjacent distal segment by compressing at least one of thesegment containing the preselected component and a segment containing areagent distal of that segment, thereby opening a breakable seal andeither propelling the reagent into the segment containing thepreselected component or propelling the preselected component into thesegment containing the reagent.